The attachment of bifunctional analogues of the strong metal chelating compound EDTA (ethylene diamine tetraacetic acid) by a covalent bond to biological molecules is of considerable interest, since the metal complexes of such a bifunctional chelating agent can be applied for radiolabelling of small molecules, macromolecules (antigens, antibodies), membranes, cells, in radioimmunoassays, and as tumor locating reagents.
Meares et al (M. W. Sundberg, C. F. Meares, D. A. Goodwin and C. I. Diamanti, J. Med. Chem., Vol 17, 1304, 1974) prepared 1-(p-aminophenyl) ethylenediaminetetraacetic acid (1) and bound it by diazotization to human serum albumin and bovine fibrinogen. They also used the bromoacetyl derivative bound to bleomycin as a reagent in tumor localization (Meares et al., J. Med. Chem., 22, 1019, 1979; J. Nucl. Med., 22, 787, 1981).
The big disadvantage of Meare's compound 1 is in the low and not always reproducible results, due to the sensitivity of the p-nitrophenylene diamine system, which tends to undergo, under the alkylation conditions, side reactions (W. Henhorf and C. K. Ingold, J. Chem. Soc., 1009, 1927).
Yeh, et al (S. M. Yeh, D. G. Sherman and C. F. Meares, Anal. Biochem., 100, 152, 1979) prepared a series of substituted EDTA (2) through amino acid conversion via borane reduction of amides and alkylation with bromoacetic acid. This series of compounds lacks the amino function needed for diazotization and requires additional protection of the EDTA function during the coupling process to the biological macromolecules.
Other EDTA analogues have been attached to monoclonal antibodies and used for detection of mouse erythroid tumors using a high resolution gamma camera. The chelatable radioactive metals with short half-lives, gallium-67, indium-111 and technetium-99 m are optimal for gamma camera imaging, while gallium-68 is optimum for positron emmision tomography, and scandium-47 or other alpha emitting isotopes are optimum for therapeutic effects (D. A. Scheinberg, M. Strand and O. A. Gansow, Science, 215, 1511, 1982).
The chelates tried by Scheinberg et al., included 1-(p-benzyldiazonium) EDTA (3), the p-hydroxybenzimidate of (3), 1-(p-carboxymethoxy benzyl) EDTA (4) and carboxycarbonic anhydride of DPTA diethylenetriaminepenta acetic acid) (5).
From all the EDTA analogues described above "only 4 could be conjugated to immunoglobulines in sufficient yield without large losses of biological activity" (quotation from Scheinberg et al).
Other studies in similar directions are labelling of preformed liposomes with Ga-67 and Te-99 m, using DPTA-anhydride (D. Y. Hnatowich, B. Friedman, B. Clancy and M. Novak, J. Nucl. Med., 22, 810, 1981) which was previously reported by W. C. Eckelman, S. M. Karesh and R. C. Reba, J. Pharmac. Sci., 64, 704, 1975).
Evaluation of the viability of In-111 labelled DPTA, coupled to Fibrinogen, was also reported by Hnatowich et al (W. W. Sayne, D. Y. Hnatowich, D. W. Doherty, R. L. Childs, D. Lanteigne and Y. Ansell, J. Nucl. Med. 23, 627, 1982).
The above examples show that the common approach to the synthesis of EDTA analogues has been the reduction of the appropriate nitrile or amide and subsequent alkylation.
From this lengthy introduction it can be surmised that new bifunctional chelating agents incorporating a strong metal chelating center (the EDTA type center), and a second functionality, sterically well separated from the EDTA center, which in addition are stable under the common conjugating conditions, will be of great interest for use in radioimmunoassays and for tumor imaging. The compounds described in this application are novel and correspond to those requirements.